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THE EFFECTS OF CANDIDA ALBICANS ON THE 
ESOPHAGOGASTRIC AREA OF THE PORCINE STOMACH 



by 



HOWARD ROBERT BIXBY 

B. S., Kansas State University, 1959 
D. V. M., Kansas State University, 1961 



A MASTER'S THESIS 



submitted In partial fulfillment of the 

requirements for the degree 

MASTER OF SCIENCE 

Department of Pathology 

KANSAS STATE UNIVERSITY 
Manhattan, Kansas 

1964 

Approved byt 



^pprov^ byt 



Major Professor 




LD 

C 2. 

. Djct^s-ne^^-K table of contents 

INTRODUCTION . 

REVIEW OF LITERATURE , ^^^ ^^ ; 

MATERIALS AND METfKa)S , ,^^^ g 

Survey Specimens ^ 

Collecting and Processing •••••.........., 6 

Gross Pathology ^^^ ^ 

Grades of Keratinization 7 

Hycology ,,,, 7 

Histopathology ......♦...,, a 

Controlled Experiment , g 

History of Animals Used »*.,.,., 3 

Experimental Design o 

Fecal Examination for Candida Species ♦,,, 10 

Blood Studies , , jj 

Necropsy Data , .. 

Mycology ^2 

Histopathology ,,, , 12 

RESULTS 

12 

Survey Specimens ,,,, *» 

Gross Pathology , ^^ ,. 

Mycology , , ^^^^ . 

Histopathology ^ ^ ^ ^ .^ 



Hi 



Controlled Experiment *• «... , ,, 17 

Fecal Examination for Candida Species 17 

Blood Studies ****»«, .t^.. , ....,...,., 18 

Necropsy Data ......,,, I8 

Mycology , , .......,,,»,,,,,, 20 

Histopathology •..••.. ....•...,..,,.,, 20 

DISCUSSION 22 

ACKNOWLEDGMENTS 25 



LITERATURE CITED 



26 



INTRODUCTION 

Gastric ulcers have long been known to occur in the glandular 
portion of the porcine stomach. Many times these ulcers accompanied 
diseases causing chronic gastroenteritis. Ulceration of the glandless 
esophageal portion was described by Bullard (1951). Since that time, 
esophagogastric ulcers have been reported with increasing frequency. 

Losses from gastric ulcers have been reported at the various swint 
testing stations throughout the country. Presently, field cases are 
appearing in widely separated herds with regularity. In Kansas, losses 
have varied froa one pig In a herd to 2(U mortality rate within a five 
day period. 

The cause of esophagogastric ulcers has been attributed to many 
entities. Various chemical el«nents and antibiotics used as feed addi- 
tives have been incriminated. Environmental conditions and diet have been 
suggested as playing a role in the etiology. The yeast-like fungus, 
Candida albicans , has been considered to have a keratinolytic or 
keratinogenic effect on the esophagogastric epithelium. The etiology 
and the pathogenesis are, at best, incompletely understood* 

This experiment was designed to study the effects of C. albicans 
on the esophagogastric area of the stomach. Mycologic findings were 
correlated with the gross and histopathologic data. 

REVIEW OF LITERATURE 

Bullard (1951) published the first specific article on esophago- 
gastric ulcers. Upon necropsy of a 500 pound boar, the only lesion found 



was a large round uXcei completely surrounding the esophageal orifice. 
The animal was anemic, and there was blood throughout the small and 
large intestine. Few succeeding references can be found on this subject 
until 1959. The possibility that a new condition had originated Is 
probable. However, there is a remote possibility that this condition 
has gone unnoticed for some time. 

An alimentary mycotic condition involving pigs on an excessive or 
prolonged intake of antibiotics was described by Quinn (1952). The area 
of Involvement was from the esophagus to the large Intestine. In one 
pig the cardia was occluded by an inflammatory fungal lesion which 
yielded pure cultures of Monilia ( Candida ). Losses dropped when anti- 
biotics were withheld. 

Kowalczyk (1958) reported 6 cases of stomach ulcers. Some pigs 
were housed In groups of 5 to 7, while others were in a group of 50 kept 
outdoors. A balanced ration was fed. 

In Great Britain an outbreak of mucormycosis was observed by Gitter 
and Austwlck (1959) in pigs that were two days to 13 days old. C 
^^^^<'3"» was found concurrently with Rhlzopus raicrosporus . It was noted 
that C. albicans only invaded the stratified squamous epithelium of the 
stomach, esophagus, mouth, and tongue. R. mlcrosporus invaded only the 
glandular parts of the Intestinal tract. Lesions attributed to candidl- 
asls were characterized by an excessive amount of mucus and desquamated 
cells found on the surface of the epithelium. The stratified squamous 
epithelium showed degenerative changes from the invasion of yeast cells 
and pseudomycelia. Congestion of the blood vessels In the submucosa was 
also noted. 



\- '■• 



9 



Kowalczyk et al, (i960) were able to demonstrate stomach ulcers by 
gastroscopic examination. Most were located in the esophagogastric are* 
of the stomach. In early stages, small wartollke proliferations of the 
stratified squamous were observed. These areas would slough, leaving 
small, dry, rough areas* As the process continued, the small areas 
would coalesce with others* As the areas increased in size, they also 
became deeper. When erosion of an artery took place varying amounts of 
hemorrhage occurred. Kowalczyk et al . (1960) concluded that stomach 
ulcers were not uncommon in swine. Over a three year period, they re- 
corded 12 deaths in 161 swine. There was no apparent relationship of 
gastric ulcers to dietary supplements of zinc, copper and cadmium. How- 
ever, there was an indication that a seasonal factor was Involved. 

Osborne et al.. (1960) observed a Candida infection In a group of 
140 artificially reared pigs. Ninety-three lived to 8 weeks of age* 
Lesions of candidiasis were observed In 25 of the 47 that died* Yeast 
cells and pseudohyphae were mixed with desquamated epithelium and neu- 
trophils. The organisms demonstrated were usually In the superficial 
layers of the epithelium. Occasionally the pseudohyphae would penetrate 
the mucosa, but the cellular reaction was the same as the areas that con- 
tained no pseudohyphae. It was suggested that a low-grade toxemia pro- 
duced by Escherichia coll , strain 0K57, may have been a predisposing 
factor. These workers also noted candidiasis was primarily present In 
pigs on a diet containing large amounts of sugar. 

Esophagogastric ulcers occurred in twine at the boar testing stations 
In Iowa. Berg (i960) suggested that management practices, confinement. 



'-r .■ ■ *-' •**. 



and high energy-low fiber ration may have been the cause of these lesions. 

In England, Buntain (1961) reported deaths due to esophagogastric 
ulcers in pigs on a nutritional experiment. Various trace elements were 
added to the diet in large quantities. The deaths were almost entirely 
limited to the groups fed large amounts of copper; however, these results 
could not be repeated. It was considered that copper plus an unknown 
toxic factor was the causative factor. 

Thoonen and Hoorens (1961) in Belgium, found a b% incidence of 
gastric ulcers in 600 pigs presented for post-mortem examination during 
a two-year period. Most ulcers were in the region of the cardia. Of 
the 29 ulcers observed, 10 were positive for Geotrichum ox Candida . In 
all cases, lesions of other diseases were found. They found no relation- 
ship of "molds", feed supplements, or parakeratosis in gattrlc ulcertt 

In two experiments Perry et al . (1963) produced esophagogastric 
ulcers by feeding galatinized corn (heat treated). In the first experiment, 
the ulcers were responsible for a 39?i mortality rate. The pigs receiving 
gelatinized corn in the second experiment had a 52^ incidence of ulcers, 
and the mortality rate was 11$. No ulcers were observed in control groups 
receiving raw yellow corn. 

Grlffing (1963) examined 610 pigs from six different sources. His 
results suggested that keratinization contributed to the pathogenesis of 
esophagogastric ulcers. Bacteriologic and mycologic studies revealed 
that only C. albicans could be Isolated routinely from the ulcers or 
denuded epithelium. 

C. albicans was isolated from eight herds in northwestern Wisconsin 
which were encountering heavy death losses. Baker and Cadman (1963) ' 



isolated the organism from the esophageal region of the stomach. Ulcer- 
ative and inflammatory lesions were also reported in this region* 

Rothenbacher et a^.* (1963) described cardiac ulcers in Michigan 
pigs. They found no relation of this type of ulcer to the peptic ulcer 
commonly associated with the glandular areas of the stomach. A relation- 
ship of the parakeratotic proliferative changes in the erosion and ulcer- 
ation existed. These workers supported the theory that some toxic agent 
probably found in the feed was responsible for the pathogenesis of esophago- 
gastric ulcers. 

Swine from the Indiana Swine Evaluation Station were observed by 
Curtln et a^. (1963). Of 443 animals observed, 87 showed evidence of 
ulcers. C. albicans was consistently isolated from the lesions. Pteudo- 
hyphae and yeast cells were often observed on histopathologic examination. 
Rapid growth of the pigs appeared to be important to the genesis of the 
esophagogastric ulcers. A seasonal occurrence also was noted* 

Blaxland and Markson (1954) made some interesting observations on 
candidiasis in the turkey* Antibiotics in feed were thought to be a 
contributing factor. Malnutrition or other external factors were sus- 
pected of playing an important role in candidiasis. These authors felt 
that increased virulence of the fungus is a possibility that should not 
b« overlooked. 

The keratinolytlc properties of C. albicans In the presence of 
glucose was demonstrated by Kapica and Blank (1957). 

Numerous referwices Indicate C. albicans is commonly isolated from 
esophagogastric ulcers. Further studies are warranted in order to 









determine the specific role of C. albicans in the pathogenesis of , . 

■J esophagogastric ulcers* 

'S : MATERIALS kW METHODS 

Survey Specimens 

Collecting and Processing . Specimens were collected at a Kansas City, 
Kansas abattoir. For the first 30 minutes of each hour, stomachs were 
collected from a moving line during a five-hour period. During this time, 
270 specimeni were selected from approximately 2,200 (12.33^) pigs which 
: weighed 180 to 220 pounds. It was estimated that the specimens obtained 
originated from at least 25 different sources. 

A gross examination was made of the esophagogastric region, which 
was removed from the glandular portion, and cut Into two parts. An atten^it 
was made to obtain representative tissue on each of the two portions as 
determined by the gross examination. One part of the sample was tagged 
for later Identification and fixed In 10^ formalin which was changed 
periodically. The other part was placed In a labeled plastic bag, and 
temporarily put In a freezer box containing dry Ice until these specimens 
could be stored at -20 C. 

Gross Pathology . Studies of gross pathology were confined to the 
esophagogastric area. Denudation of the esophagogastric epithelium was 
measured as to the area of Involvement and the depth of erosion. Hemorrhagic 
areas were considered evidence of subepithelial Involvement. All observa- 
tions were recorded as to type, location, and size. 



/. 

Grades of Keratlnlzation . The classification evaluation of amount 
of keratinization varied slightly fiom those employed by Griffing (1963)» 
The following scale was usedt 

Grade Kq - No keiatinization visible grossly 

Grade Kj - Very slight keratinization 

Grade K2 - Slight keratinization 

Grade IC - Moderate keratinization 

Grade K^ - Marked keratinization 
Grades of keratinization were determined grossly and recorded for the 270 
stomachs. 

Mycology * Sabouraud's dextrose agar was used to culture the tissues 
stored at -20 C. Chloramphenicol (0.05 mg./ml.) and cycioheximide^ (0.5 mg./ 
ml.) were used to inhibit bacterial and mold contamination. Pieces of tissue* 
0.5 sq. cm. in size» were embedded in the agar. Six tubes of this medium were 
used for each specimen. Four tubes were incubated at 25 C, and two were 
incubated at 37 C. The tubes were examined daily for seven days, then twict 
a week thereafter. All culture tubes were observed for 30 days. All colonies 
appearing to be yeasts were Gram-stained. Colonies characterized by aerial 
hyphae were stained with lacto-phenol cotton blue. Upon microscopic examina- 
tion, the stained preparations were Identified as to genus when possible. 
The imperfect fungi were classified according to Barnett (i960). With the 
exception of the yeast-like organisms, no further identification was at- 
tej^ted. The yeast-like organisms characterized by being Gram-positive, 



1 Difco Laboratories, Detroit, Mich. 

2 Actidone, Upjohn Co., Kalamazoo, Mich. 



oval, budding, thin walled cells (2-5 microns) were puxifiad as described 
by Laskowski et al_. (i960). Confirmation of Candida spp. and identifica- 
tion of C. albicans was accomplished by the use of corn meal agar^, Levine 
EMB agar , and sugar fermentation tests. Corn meal agar and Levine EMB 
agar were used as described by Griffing (1963). The procedure outlined 
by Laskowski et al. (i960) was used for the fermentation tests. By cor- 
relating the results of the various tests utilized, C. albicans could be 
positively identified. 

Histopatholoqy. Tissues collected for histopathological examination 
were fixed in 10^ buffered formalin. Hematoxylin and eosin were used as 
the tissue stain, and Gridley's stain was employed to demonstrate fungi. 

Controlled Experiment 

History of Animals Used . The pigs selected for this experiment 
originated from three litters born and reared at the Kansas State University 
Veterinary Research Laboratory. Seven days separated the oldest and youngest 
litter. At approximately four weeks of age, the pigs were given access to 
a commercially prepared, pelleted ration free of antibiotics and considered 
to have all known nutrients present. This ration was utilized throughout 
the experiment, and after weaning, was the only nutritional source. 

At approximately six weeks of age, the pigs were vaccinated with 
modified-live hog cholera virus . Anti-hog cholera serum^ was given 



1 Page's Complete Hog Feed, Manhattan Milling Co., Manhattan, Kans. 

2 Alocine, Haver-Lockhart Laboratories, Kansas City, Mo. 
^ Armour Pharmaceutical Co., Kankakee, 111. 



simultaneously. These animals were also vaccinated with erysipelas 
bacterin. - . ._ 

Experimental Design . The pigs were divided into three groups. The 
selection was based upon weight, sex, and litter of origin. Group one was 
designated as the experimental group, group two as the surgical control 
group, and group three as the dietary control group. ' ' ' '-: -*' ''", 

Gastrotomies were performed on the pigs in group one. The esophago- 
gastric epithelium was classified as to the amount of keratinization and 
denudation. The area was swabbed for culture purposes, mildly scarified, 
and innoculated with C. albicans . A 48-hour Sabouraud*s dextrose agar 
culture of C. albicans suspended in 3 ml. of physiologic saline was used. 
Another swab was saturated with this suspension, and applied to the area. 

At weekly intervals, for the first two months of the study, 40 ml. 
of physiologic saline with 50 million C. albicans cells per ml. wera 
given by stomach tube. During the third and final month this dosage was 
given twice weekly to those pigs remaining on the experiment. The stomach 
tube was inserted into the esophagus to the thoracic inlet. Care was taken 
not to allow the stomach tube to enter the stomach. 

Group two was subjected to the same surgical procedures and the 
esophagogastric epithelium was Classified as to the degree of keratinization 
and denudation as in group one, except sterile physiologic saline Kas swabbed 
into the scarified area of the stomach. Forty ml. of physiologic saline 
was given by stomach tube at the same time group one was given C. albicans 



1 Rhusigen, Pitman-Moore Co. Div., Allied Uboratorles, Inc., 
Indianapolis, Ind. 



10 



and saline* 

Group three was not subjected to surgery or to the use of the stomach 






tube. This group received the same diet as groups one and two. 

Pentobarbital sodium was the anesthetic used for the surgery in 
groups one and two. 

Saline suspensions of C. albicans cells were counted by the Coulter 

2 
electronic particle counter and diluted to the desired concentration. 

Nineteen pigs were to be used for this experiment; however, 2 pigs 
died during surgery* Of the remaining 17, there were 6 pigs in group one, 
6 in group two, and 5 in group three. 

Fecal Examination for Candida Species. Fecal specimens were taken 
three times before the experiment started and once a week during the 
experiment for each individual pig until each was euthanatized. These 
specimens were labeled and frozen at -20 C. until cultured. The time 

in frozen storage varied from one week to three months. Pagano Levine 

3 
medium was used as the selective medium for the determination of Candida 

4 
species* Ten mg* of 2,3,5-triphenyl-tetrazolium chloride and 50 mg. of 

neomycin were added to each liter of Pagano Levine medium. This medium 

was considered to be selective for the Candida species. The principle of 

this test as described by Pagano sL 3l* (1957-58) is based upon the different 

capacities of Candida species to reduce T.T.C. and produce varying degrees 

of color. 

1 Halatol, Jensen-Salsbery Laboratories, Inc., Kansas City, Ito* 

2 Coulter Electronics, Inc., Hialeah, Fla. 

3 Difco Laboratories, Detroit, Mich* 

4 T.T.C, Difco Laboratories, Detroit, Mich. 



11 



Pagano Levlne medium was used as a quantitative test for Candida 
species as employed by Coles (1963). The fecal specimens stored at 
-20 C. were weighed aseptically into one-gram portions, and diluted in 
9 ml. of sterile saline. One tenth and 1 ml. of thoroughly mixed sus- 
pension was placed into sterile petri dishes. Approximately 15 ml. of 
Pagano Levine medium was poured in each plate and mixed thoroughly. Dupli- 
cate plates were run on each dilution. After incubating for 48 hours at 
37 C, smooth, round, glistening colonies with a cream to light pink color 
were counted. By this method, the relative number of Candida species In 
the feces could be determined. Periodic gram stains of colonies counted 
were made. The inhibitors in the medium were considered to be functioning 
properly when cells with the morphology characteristic of Candida species 
were obtained. 

Blood Studies . Blood samples were taken three times before the 
onset of the experiment and once every two weeks thereafter. Samples 
were also taken Immediately before necropsy. Disodium ethylenedlamlne- 
tetraaeetate was used as an anticoagulant. The following tests were runt 
packed cell volume, hemoglobin, total WBC and differential WBC» 

Necropsy Data . Pigs were randomly selected from each group for 
euthanasia at one month, two months, and three months after the beginning 
of the experiment (Table 1). A complete gross examination was made. ; "; 
Tissues were taken from all major organs, and fixed In 10^ buffered 
formalin. Brain and kidney were taken aseptically and frozen at -20 C. 
for mycologlc studies. Conant e^ ^. (1954) rqsorted Isolations of Candida 



^ EDTA, Cambridge Chemical Products, Inc., Dearborn, Mich. 



12 



species In these organs. The esophagogastric area of the stomach was 
observed and graded as to keratinization and amount of epithelial denuda- 
tion. This tissue was collected for histopathologic and mycologic studie* 
in the same manner described for collecting and processing the survey 
specimens. 



TABLE l—The Number of the Pigs Necropsied for Each Group During the 
Experiment 



1 month 2 months 3 months 
^oup after surgery after surgery after surgery 
(pigNo.) (pig No.) (pig No.) 



» ' • • 33 13, 28, 26 18, 35 

* 23 , 12, 17, 27 26, 31 

• 25 32, 34 11, 21 



J' 
Mycology. The same methods were employed for both culturing and 

identification as was described under survey specimens. The brain, kidney 
and esophagogastric epithelium were cultured. 

Histopatholoav. Tissues from all major organs were saved for histo- 
pathologic studies. The esophagogastric areas of the stomach was treated 
in the same manner as described under histopathology of the survey specimens. 

RESULTS 

Survey Specimens 

Gross Pathology. The 270 survey stomachs were classified as denuded 
or not denuded. There were 219 (81.1^) that showed no denudation, and 51 



u 



(18.9^) that showed denudation. Sub-epithelial petechiation was observed 
in 22 (8.1%) of the specimens and these were recorded as severe denudation. 
No ulcers wer« observed. 

Hid amount of keratinization was categorized and classified. The 
keratin layer was considered by Smith and Jones (1961) to be the most super- 
ficial layer of stratified squamous epithelium. They stated that it may b* 
considered abnormal if present in excessive amounts or in abnormal places. 
Seventy-seven (28.5^) of the survey stomachs showed no keratinization, 
while 193 (71.5^) showed varying amounts of keratinization. Some stomachs 
had excessive amounts of keratinized tissue over the entire esophageal area. 
Others had scattered wart-like projections at various places throughout 
with no or varying amounts of keratinization on the remaining tissue. In 
the latter case, only the wart-like projections were considered for keratin- 
ization classification. According to the grading used, there were 77 (28.5Jt) 
classified as Kq, 73 (273^) as Kp 64 (23.75^) as K^, 43 (15.9^) as K3, and 
13 (4.8^) as K^. ^ '. 

The number of denuded stomachs for each grade of keratinization wat 
determined (Table 2). For each grade increase in keratinization, denuda- 
tion increased from 10.6^ to 16.4St;. The "row x column chi square test for 
proportionality" (Snedecor, 1956) was employed to test the increase in pro- 
portion (Table 3). At the 0.05 level, the grade K^ was shown to have a 
significant increase in proportion of denudation than in grade IC. The 
same statement could be made concerning the other groups having keratiniza- 
tion when comparing them to Kq. The results of this chi square test would 
indicate there is a relationship of keratinization to denudation, but the 
degree of keratinization is not necessarily a factor in this group. 



M 



TABLE 2— Number of Denuded Stomachs In Each Keratinization Groupings 



Keratini- 

zation 

grade 


No. In 

each 
group 


Denudat 
No. 


Ion 


present 
% 


Severely denuded 

No. % 


h 


7f 


I 




1.4 


• d 


h 


n 


10 




13.7 


1 6.8 


h 


M 


17 




26.6 


6 9.4 


H 


4S 


16 




37.2 


• 18.6 


h 


la 


T 




53.3 


t 23.1 



Totals fS$ 

Total % denudation 



51 



18.9 



8.1 



2 
TABLE 3— Results of Row x Column Chi Test of Proportionality. 

= -0?S Rp1«»et wiihon r.hl^ 'J.fU 



.05 Reject when chi^ 3.84 



Hgt 


Chi^ 
values 


Conclusion 


V 


Chi^ 
values 


Conclusion 


■^ = •^1 


8.30 


reject 


h = ^2 


3.76 


accept 


Ko = K2 


20.85 


reject 


K2*K3 


1.25 


accept 


^0 = ^3 


29.90 


reject 


K3=i^ 


0.60 


accept 


h'h 


44.60 


reject 









Mycology . Of the 270 stomachs collected, 50 stomachs were selected 
randomly for mycological studies. The amount of keratinization and denuda- 
tion is representative of the 270 observed on gross examination. 



15 



There were a variety of organisms isolated as shown in Table 4. With 
the exception of Candida and Trichosporon species, the fungal isolations 
were identified upon initial isolation by colony and microscopic morphologic 
characteristics. Trichosporon was identified on the special media used to 
identify the Candida species. 



TABLE 4— A Listing of the Various Organisms Cultured During Mycological 
Studies from Esophagogastric Area 



Genera 


No. 
isolated 




Genera 


isolated 


Streotomvces species 


21 




Tfichosporon species 


i 


Geotrichun species 


9 




Aspergillus species 


2 


Candida species 


8 




fJtycelia sterila 


S 


Penicillium species 


3 








* Two isolates were 


identified 


as C. 


albicans. 


ib 



Candida species and C. albicans were primarily considered in this 
survey. There were 8 (16^) Candida species isolated. Two {a%) were later 
identified as C. albicans . 

Of the 3 Candida isolates, an inconsistency existed between the isola- 
tion of the organism and the degree of keratinization present (Table 5). 
In comparing the Candida species cultured to the amounts of keratinization 
present, 3 were isolated from 17 specimens classified as Kq, 3 from 11 
classified as Ki, and 2 of 6 from Kg. No isolates were made from the K2 



u 



on K. stomachs (Table 5). Denudation was observed in one stomach from 
which a Candida isolate was made. It was graded as K3. 

Two isolates were C. albicans . The kexatinization grades were K_ 
and IC. * No denudation was noted in these two specimens* 

TABLE 5— Summary of the Relationship of Keratinization and Epithelial 
Denudation to the Incidence of Candida Species 



















Kexat: 


Lnization 


Denuded 
epithelium 
No. % 




Incidence of Candida 
species 




grade 


No. 


% 


My CO logic 


Histopathologic 
(Gridley's) 


Total 


h 


17 


34 


I 


2 


3* 


1 


3»* 


h 


11 


22 


9 


9 


3* 


1 


4 


^ 


13 


26 


a 


4 


e 


1 


I 


% 


6 


12 


9 


6 


2 





t 


% 


3 


6 


2 


4 


9 





• 


Total 


50 


- 


8 


16 


• 


3 


Ml 


* Includ 


es one 


isolate 


of C. 


albicans; no denudation observed. 





** One positive for both methods employed. 



Histopatholoqy . Microscopic examination was made of the same 50 
specimens utilized for mycologic examinations. Sections of the stomachs 
were stained with haematoxylin and eosin, and Gridley*s stain. 

The stratified squamous epithelium of the esophageal region was often 
characterized by accumulation of keratin on the surface. Nuclear retention 
was observed in the keratin layer. A layer of large, clear cells was 
observed in the epithelium of many specimens which did not stain with the 
methods anployed. No significance was attached to these cells. 



'^i^:-' 



17 



Forty-one of the 50 tissues examined microscopically appeared normal. 
The rete pegs were normal in length, and evidence of inflammatory cells ot 
vascular changes were not observed. 

Denuded epithelium was noted on six stomachs in which the lamina 
propria was exposed. A mild infiltrative process characterized by an 
increase in eosinophils, neutrophils, and lymphocytes was observed in four 
of the six specimens. In these six specimens, basal cell proliferation 
MS evidenced by the downward extension of the rete pegs. 

Three of the tissues observed had inflammatory changes in the propria; 
however, no pathologic changes were seen in the stratified squamous epithe- 
lium. 

Pseudohyphae and budding yeast cells were observed in three of the 50 
sections stained by Gridley's technic and were tentatively identified at 
Candida species. Ajello et al^. (not dated) considers the finding of 
pseudohyphae and budding yeast cells simultaneously present in tissue to 
be diagnostic of Candida species. 

Controlled Experiment 

Fecal Examination for Candida Species . Fecal specimens were taken one 
week previous to the beginning of the experiment and at regular intervait 
during the experiment. During the first two months of the experiment, 
the fecal specimens were collected seven days after the animals In group 
one were given C. albicans , and during the third month, the specimens were 
collected four days after the animals in group one were given C. albicans . 



» 



No apparent increase of Candida species was observed in the C. albicans 
group over the control groups not receiving C. albicans during the first 
60 days of the experiment. During the final 30 days of the experiment, 
all specimens from group one were positive for Candida species; however, 
in group three, 703^ of the samples taken were positive (Table 6). 

Blood Studies. Packed cell volume, hemoglobin, total WBC and WBC 
differential were run for each pig on three blood samples before the 
experiment and every two weeks during the experiment. All findings were 
considered to be in the normal range for swine. Data compiled for in- 
dividuals and as group means showed no significant difference between 
the groups. 

Necropsy Data. Pigs were necropsied at various times during the 
experiment (Table 1). There were no gross lesions or disease processes 
observed at necropsy which were considered to have affected the outcome 
of the experiment. 

A careful examination of the esophagogastric area revealed pathologic 
changes which were not confined to any one group. All degrees of keratini- 
zation were observed, irrespective of the groups from which they were de- 
rived. Denudation was present in 5 of the 6 pigs in group one, 5 of the 
6 pigs in group two, and 3 of the 5 pigs in group three (Table 7). Sub- 
epithelial hemorrhage was observed in 1 pig from group one, 2 from group 
two, and 1 from group three. 

An ulcer was observed from pig No. 36 in group one. The lesion was 
1 cm. X 2 cm. in size. The edges were slightly elevated, and the base was 
brown and rough. A similar lesion was observed from pig No. 17 in group 



If 



TABLE 7— Gross Observations of the Esophageal Area of the Stomach at 
the Time of Surgery and Necropsy 



Pig 


Keratinization 




Denudation 


Ulceration 


No. 












Surgery 


Necropsy 




Surgery Necropsy 


(Necropsy) 








Group 


1 




33 


Kt 


^ 









13 


% 


% 




* . ' ♦ ' ' 





23 


.. % 


^4 




© '♦ 





36 


% 


h 




♦ 


, 4 


18 


1^ 


K3 




♦ ♦ 





33 


H 


% 




e e 




i '- 








Group 


2 




23 


H 


«5| 




4 





12 


H 


h 




t ^ ♦ ' 





17 


^ 


Ka 




• t 


+ 


27 


H 


«^ 




. ' 4 ' 





26 


% 


i^ 




♦ .♦ 


0* ■ 


31 


h 


»^ 


Group 


e 

3 





25 


• 


^ 




^ 1 . 

4 


d 


32 


• 


H 




#'■ 





34 


- 


U 










11 


m 


H 




4 





21 


m 


H 




♦ 


t 



* Small, white, glistening area, considered to be a healed ulcer. 



20 



two which was 1 cm. in diameter. Pig No. 26 in group two •xhibited a 
small, white, glistening area 0.5 cm. in diameter which was considered 
to be a healed ulcer (Table 7). 

Mycology . At the time of surgery, the esophagogastric epithelium was 
swabbed for culture purposes on those pigs subjected to surgery. Identifi- 
cation of Candida species and C. albicans were carried out. In group one, 
two Candida species plus one C. albicans were found. In group two, one 
Candida species plus three C. albicans were identified (Table 8). 

At necropsy, mycologic examination of the esophagogastric area re- 
vealed the presence of six C. albicans » four were from group one, one 
from group two, and one from group three. Five other Candida species, 
not C. albicans , were isolated (Table 8). These Candida species were 
found in all 6 animals of group one, 4 of 6 in group two, and in 4 of 5 
from group three. 

Kidneys and brains of all animals were cultured, and no isolations 
were made. 

Geotrichum species was isolated from eight swabs taken at the time 
of surgery. At necropsy, three isolates of Geotrichum species were made. 

HistopatholoQv. Tissues collected for histopathologic examination 
were stained with Gridley's stain and examined. Four observations of 
pseudohyphae and budding yeast cells were observed. As previously stated, 
these were tentatively considered as Candida species. These cells were 
found in the outermost portion of the keratin layer. Only one of these 
was positive by the culture method (Table 8). Sections stained with 
haematoxylin and eosin from each stomach were examined. Uniform findings ' 



21 



TABLE 8— "■Summaiy of the Mycologic Studies at the Time of Surgery and 
Necropsy (Gridley's Stain Included) 





Time of 
Myco 


Surgery 
loqic 




Time of Necropsy 




Pig 


Mycoloqic Gridley's* 


Total 


No. 


C. 
albicans 


Candida 
species 


C. 
albicans 


Candida Candida 
species species 


Candida 
species 




, 


. •^■3^'-. „■ 


Group 1 




.. 


33 





■ ^ • ■>. 


■• 


+ .. : f. .: 


♦ ■" ' 


^ 





■ :-♦ . 


♦ 


i- 


+ 


28 








d 


+ -^ : . 


+ 


36 





• 


♦ 


■ -♦■ , it- ■ ■. 


. . #; .■ 


18 


+ 


-: *: 


♦ 


+ . . • 


♦ 


35 





♦ 


Group 2 


-*- 


♦ 


23 





♦ 





' ♦ 


♦ 


12 





• 





t 





17 


-t- 


♦ 


a 


• 





27 


+ 


♦ 


♦ 


+ ♦ 


+ 


26 





• 





-*- • 


.. ♦■ ■• 


31 


+ 


♦ 


e 

Group 3 


♦ 


+ 


25 


• 


• 


e 


+ ■ "'f 


♦^ 


32 


- 


• 





4 .. >§■ 


■f.. 


34 


- 


- 


♦ 


"*• • 


♦ 


11 


1« 


• 


e 


♦ 


♦ 


21 


- 


w 


9 








Total 


4 


7 


6 


11 4 


14 



♦ Budding yeast and pseudohyphae observed. 



were observed throughout the animals irrespective of groups* Denudation, 
as described previously, was found in all but three tissues examined* 
Infiltration of neutrophils, eosinophils, and lymphocytes was mild t© 
moderate in all specimens with exception of two* Ihs other histologic 
alteration observed was extension of rete pegs into the lamina propria. 

DISCUSSION 

Studies were conducted which showed an epidemiologic relationship 
of esophagogastric epithelial denudation and ulceration to the incidence 
of Candida species, especially C. albicans * In the survey group, there 
were no esophagogastric ulcers demonstrated and 13*9/1^ denudation observed. 
The mycologic incidence was 163^ Candida species and A% C. albicans . In 
the experimental pigs, the findings weret 11.8^ ulcers, 76.5/1^ denudation, 
82.451^ Candida species, and 35.3?^ C. albicans. When considering the 
etiology of esophagogastric ulcers, the presence of Candida species and 
£* albicans should be considered, at least as « contributing factor* 

In a similar survey, Griffing (1963), found no esophagogastric ulcers, 
9i denudation, 27?^ Candida species, and no C. albicans . The Candida species 
isolated was largely C. zeylanoides , which he considered to have no patho- 
logic significance. Disregarding C. zeylanoides , the incidences of Candida 
species as related to denudation and ulceration, are similar in the two 
surveys. The same relationship exists when comparing the incidence of 
C. albicans * 

Baker and Cadman (1963), Curtin et al . (1963) and Griffing (1963) were 
able to isolate C. albicans routinely from esophagogastric ulcers. The 



findings in this experiment further demonstrate the relationship of 
candidiasis to esophagogastric ulcers* 

When comparing the survey animals to the experimental pigs, no 
relationship as to diet was determined. The survey animals originated 
from a number of sources, and the physical and chemical characteristics 
of the feed undoubtedly varied from animal to animal, depending upon the 
source. There were no antibiotics or sugar added to the feed of the 
experimental pigs. ...... . 

Seasonal occurrence, as mentioned by Kowalczyk (i960) and Curtin 
et al . (1963) was not considered in the evaluation because all stomach 
specimens were obtained during the same season of the year. 

Berg (1960) and Osborne et al^. (i960) considered various environ- 
mental factors may be of importance to esophagogastric ulcers. It was 
felt that these factors should not be overlooked. The experimental 
groups were subjected to surgery, the collection of fecal specimens from 
the rectum, the collection of blood, and the withholding of feed. Pigs 
having a high incidence of ulcers have been derived from swine testing 
stations (Griffing, 1963; Perry et al., 1963) or experimental animals 
which have been handled or treated quite frequently (Kowalczyk, 1960| 
Buntain, 1961). Such factors may have contributed to the high incidence 
of denudation and ulceration in the experimental groups. 

In these studies, keratinization was considered as a factor in the 
genesis of esophagogastric ulcers. Denudation, which was considered 
as possible predisposing factors contributing to subsequent ulceration, 
was shown statistically to be related to keratinization. Kowalczyk (i960) 
observed erosion of the epithelium by gastroscopic methods. 



24 



It was concluded that Candida species, keratinizatlon and envlron- 
■•ntal factors contributed to the pathologic findings of esophagogastric 
area in the pigs studied* Other factors which have been stated previously 
as possible causes or contributing factors had no apparent relationship 
to the finding in this expearlaent. 

In future studies* the use of Candida -free pigs must be considered. 
Gastroscopic examination of the esophagogastric epithelium during experi- 
mentation may demonstrate valuable information necessary to determine the 
pathogenesis and etiology of esophagogastric ulcers. 



25 



ACKNOWLEDGMENTS 

The author wishes to express his gratitude to Dr. D. C. Kelley, 
Associate Professor of Pathology, for his advice and guidance through 
this course of study* Appreciation is extended to Dr* E. H. Coles, 
Head of the Department of Pathology and Dr« H. C. Mussman, Instructor 
of Pathology, who were always available for consultations* 

Gratitude is extended to Mrs* Vivian Schrepel, Medical Technologist, 
who processed the tissue sections; and to Mrs* Virginia Herren, Medical 
Technician, for assisting in the hematologic examinations* 

The author is indebted to Kansas State University for making this 
study possible; and to the Office of the Surgeon General, Department of 
the Army, for selecting him for this course of study* 



26 
LITERATURE CITED 



AjellO) Lt, Georgt L. K., Kaplan, W., and Kaufman, L. : Laboratory 
Manual for Medical Mycology, U. S. Dept. of Health, Education, and 
Welfare, Public Health Service, Communicable Disease Center, Atlanta, Ga. 
(no date). 

Baker, E. D., Codman, L. P.i Candidiasis in Ptgs in Northwestern 
Wisconsin. J.A.V.M.A. 142 (April 1, 1963)j 763-767. 

Barnett, H. L.» Illustrated Genera of Imperfect Fungi. 2nd Ed. 
Burgess Publishing Co., Minneapolis, Minn., 1960. 

Berg, N. N.» A Gastric Ulcer Condition of Swine. Iowa State Coll. 
Vet., 22 (1960): 77-78. 

Blaxland, J. D., and Markson, L. M.i Observations on the Trans- 
missibility and Pathogenesis of Candidiasis in Turkey Poults. Brit. Vet. 
J., 110, (1954): 139-145. 

Bullard, J. F.j Gastric Ulcer in a Large Boar. J.A.V.M.A., 119, 
(Aug., 1951 )i 129. 

Buntain, D.i Deaths in Pigs on a High Copper Diet. Vet. Rec., 
73, (No. 29, July, 1961 )i 707-713. 

Coles, E. H.t "Personal Communications'*. 

Conant, N. F., Smith, D. T., Baker, R. D., Calloway, J. L. , and 
Martin, D. S.i Manual of Clinical Mycology. 2nd Ed. W. B. Saunders Co., 
Philadelphia, Pa., 1954. 

t 

Curtin, T. M. , Goetsch, G. D., and Hollandbeck, R. Clinical and 
Pathologic Characterization of Esophagogastric Ulcers in Swine. J.A.V.M.A., 
Vol. 137 (Oct. 15, 1963)i 854-860. 

Gitter, M., and Austwick, P. K. C.i Mucormycosis and Moniliasis in 
a Litter of Suckling Pigs. Vet. Rec, 71, (I959)i 6. 

Griffing, J. G.: A Study of the Etiology and Pathology of Gastric 
Ulcers in Swine. Kansas rtate University, Ph.D. Dissertation (1963). 

Kapica, L. , and Blank, F.» Growth of Candida albicans on Keratin as 
Sole Source of Nitrogen. Dermatologica, 115, (1957)j 81-105. 

Kowalozyk, T.t Stomach Ulcers in Swine. Vet. Sci. News, 12, (1958): 17. 

Kowalczyk, T., Hoekstra, W. G., Puestow, K. L., Smith, I. D., and 
Grummer, R. H.: Stomach Ulcers in Swine. J.A.V.M.A., 137, (Sept., 1960): 
339-344. 



. »**■ . 



It 



Laskowski, R. F., Barrett, J. T., Duffet, N. D., Moore, M. , and 
Sato, P. J.t Basic Techniques and New Concepts in Clinical Diagnostic 
Medical Mycology. Catholic Hospital Assn., St. Louis, iMo., Oct., 1960. 

Osborne, A. D., McCrea, M. R., and I'tenners, M. J.i Moniliasis in 
Artificially Reared Pigs and its Treatment with Nystatin. Vet. Rec, 72, 
(1960) I 237-241. 

Perry, T. W., Jimenez, A. A., Shively, J. E., Curtin, T. M., Pickett, 
R. A., and Beeson, K. M.t Incidence of Gastric Ulcers in Swine. Science, 
139 (Jan., 1963)i 349. -- • 

Quinn, A. H.t Newer Problems in Swine Diseases»Control and Treatment* 
Canad. J. Conp. Med. Vet. Sci., 16, (l952)i 265-270. 

Rothenbacher, H., Nelson, L. W., and Ellis, D. J. The Stomach Ulcer - 
Gastrorrhagia Syndrome in Michigan Pigs. Vet. Med. (Oct., 1963}t 806-816. 

Smith, H. A., and Jones, T. C: Veterinary Pathology. 2nd ed. Lea 
and Febiger, Philadelphia, Pa., 1961. 

Snedecor, G. W.t Statistical Methods. 5th ed. The Iowa State 
University Press, Ames, Iowa, 1956. 

Thoonen, J., and Hoorens, J.i Maagulcera In De Pars Oesophagea 
Bij Varkens. Vlaams Diergeneeskundig Tijdschrift, 30, (l961)i 79-92. 
Cited in Griffing (1963). 



THE EFFECTS OF CA^DIDA ALBICANS ON THE 
ESOPHAGOGASTRIC AREA OF THE PORCINE STOMACH 



HOWARD ROBERT BIXBY 

B. S., Kansas State University, 1959 
D. V. M., Kansas State University, 1961 



AN ABSTRACT OF A MASTER ♦S THESIS 



submitted in partial fulfillment of th« 



requirements for the degree 



MASTER OF SCIENCE 



Department of Pathology 



KANSAS STATE UNIVERSITY 
Manhattan, Kansas 



1964 






;v»^ ^ , ■•_ *- -'^■-^'?, 



t' 



In a randomized sample of 270 swine stomachs taken during one 
day's slaughter at a Kansas City, Kansas abattoir, excessive amounts of 
keratinization of the esophagogastric epithelium was observed in a small - 
number of animals (2.9^)« 

Denudation was noted in 51 (18.9^) of the tissues observed. When 
applying the "row x column chi square test for proportionality", a 
relationship of keratinization to denudation existed, but the degree of 
keratinization was not proven to be a factor. Denudation was considered 
as possible predisposing factors contributing to subsequent ulceration. 

Fifty of the 270 tissues studied by mycologic and histopathologic 
methods revealed a 163^ incidence of Candida species and a 4^ incidence 
oi C. albicans . Forty-one stomachs were considered to be normal when 
examined histopathologically. Denuded epithelium was noted in six 2- 
tissues, and a mild infiltrative process of the proper ia was observed 

in seven tissues. 

y 
, In an experiment designed to study the effects of C. albicans on 

the esophagogastric epithelium, 8 of the 11 control animals (groups two 
and three) were infected from unknown sources throughout the experiment 
with Candida species including C. albicans . The incidence of Candida 
species in the controlled experiment was 14 to 17 (82.4^), while six of 
17 (35.^) tissues were positive for C. albicans . Gross and histopathologic 
findings revealed denudation and/or ulceration in all groups. It should 
be pointed out that six of 17 (35.:^) tissues were positive for C. albi- 
cans in the experimental groups, while only 2 of 50 (4^) were positive 
for C. albicans in the survey group. The incidence of Candida species 



in the controlled experiment was 14 of 17 (82*4;^} while the survey group 
showed 8 in 50 (16^)* This evidence indicates a possible relationship 
of Candida species to denudation and ulceration of the esophagogastric 
^itheliun* However) the surgery at the onset of the experiment) 
collecting fecal specimens from the rectum, collecting blood for study, 
and withholding feed may be factors effecting the findings in the 
" esophagogastric area. Such factors should be considered. 



•rf 



r ; .